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A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle.

Higuchi H, Iwano H, Kawai K, Ohta T, Obayashi T, Hirose K, Ito N, Yokota H, Tamura Y, Nagahata H - J. Vet. Sci. (2011)

Bottom Line: The results obtained by the simplified PCR were consistent with ones from standard PCR.This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h.The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

View Article: PubMed Central - PubMed

Affiliation: Animal Health Laboratory, Faculty of Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan. higuchi@rakuno.ac.jp

ABSTRACT
A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

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Detection of major mycoplasma mastitis pathogens by simplified (upper) and standard (lower) PCR. M. marker, 1: Mycoplasma (M.) bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).
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Figure 1: Detection of major mycoplasma mastitis pathogens by simplified (upper) and standard (lower) PCR. M. marker, 1: Mycoplasma (M.) bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).

Mentions: In this study, we compared the detection rates for seven major Mycoplasma spp. of the simplified and standard PCR assays. All ATCC strains were clearly detected by both methods (Fig. 1). Seven species of two mycoplasma strains isolated from animals with mycoplasma mastitis were also detected by both simplified and standard PCR (data not shown). Gene sequences of PCR-amplified products showed 99% homology with documented sequences in an established gene bank (intergenic spacer region). The minimum detection limit for Mycoplasma by simplified PCR was estimated to be 2.5 × 103 cfu/mL (Fig. 2). The results obtained from the simplified PCR agreed well with those from standard PCR using purified DNA from broth cultures. Our results showed that simplified PCR for detecting Mycoplasma spp. permits gene amplification without any DNA preparation and performs as well as standard PCR. In this study, we used Amdirect Plus for the simplified PCR to detect Mycoplasma spp. in broth cultures. Ampdirect Plus is a commercially available reagent used for preparing PCR samples without DNA extraction and purification [1]. We have confirmed that the use of Ampdirect Plus reduces labor and time for detecting Mycoplasma spp. in samples.


A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle.

Higuchi H, Iwano H, Kawai K, Ohta T, Obayashi T, Hirose K, Ito N, Yokota H, Tamura Y, Nagahata H - J. Vet. Sci. (2011)

Detection of major mycoplasma mastitis pathogens by simplified (upper) and standard (lower) PCR. M. marker, 1: Mycoplasma (M.) bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104175&req=5

Figure 1: Detection of major mycoplasma mastitis pathogens by simplified (upper) and standard (lower) PCR. M. marker, 1: Mycoplasma (M.) bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).
Mentions: In this study, we compared the detection rates for seven major Mycoplasma spp. of the simplified and standard PCR assays. All ATCC strains were clearly detected by both methods (Fig. 1). Seven species of two mycoplasma strains isolated from animals with mycoplasma mastitis were also detected by both simplified and standard PCR (data not shown). Gene sequences of PCR-amplified products showed 99% homology with documented sequences in an established gene bank (intergenic spacer region). The minimum detection limit for Mycoplasma by simplified PCR was estimated to be 2.5 × 103 cfu/mL (Fig. 2). The results obtained from the simplified PCR agreed well with those from standard PCR using purified DNA from broth cultures. Our results showed that simplified PCR for detecting Mycoplasma spp. permits gene amplification without any DNA preparation and performs as well as standard PCR. In this study, we used Amdirect Plus for the simplified PCR to detect Mycoplasma spp. in broth cultures. Ampdirect Plus is a commercially available reagent used for preparing PCR samples without DNA extraction and purification [1]. We have confirmed that the use of Ampdirect Plus reduces labor and time for detecting Mycoplasma spp. in samples.

Bottom Line: The results obtained by the simplified PCR were consistent with ones from standard PCR.This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h.The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

View Article: PubMed Central - PubMed

Affiliation: Animal Health Laboratory, Faculty of Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan. higuchi@rakuno.ac.jp

ABSTRACT
A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Show MeSH
Related in: MedlinePlus